GST蛋白表达、纯化步骤

GST蛋白表达、纯化步骤
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Protein Expression Protocol:

1. Transform control construct and tag-fusion-constructs into E.coli BL21(DE3) competent cell, grow overnight;

2. Inoculate single colony to 2~3 ml LB medium, and grow at 37 degree for 7~10 hours or overnight;

3. 1:100 inoculate to 3 ml LB, bacteria grow for 2-3 hours at 37 degree, then follow 1:2000 add 1 M IPTG to induce protein expression overnight at 22 degree;

4. Spin down bacteria at 12000 rpm for 1 min, wash with 1 ml ddH 2O, add 100 ul 1x PBS to resuspend the deposition, then sonicate 3~5 min on ice;

5. Spin down at 12000 rpm for 10min, separate the supernatants and deposition;

6. Boiling with loading buffer at 100 degree for 5min, Spin down at 12000 rpm for 5min, 12% SDS-PAGE;

7. After confirmed protein expressed in supernatants, increase the volume of bacteria medium. 1:100 inoculate to 100 ml LB, grow bacteria for 2-3 hours at 37 degree; then follow 1:2000 add 1 M IPTG to induce protein expression overnight at 22 degree;

8. Spin down bacteria at 8000 rpm 4 degree for 5 min, wash with 10 ml ddH2O, add 5 ml 1x PBS to resuspend the deposition, then sonicate 60 min on ice;

9. Spin down at 12000 rpm 4 degree for 10min, separate the supernatants and deposition;

10. Prepare the supernatants for purification.

蛋白表达注意事项:

1. E.coli BL21比DH5α生长要快,固体培养基上10~12小时即可长斑,液体培养基中8小时后即可生长成较大密度;切勿培养时间过长,防止菌体老化;

2. 为了后续实验的便利,应尽量减少包涵体的形成。主要有以下方法可以减少包涵体的形成: a. 降低IPTG 的浓度。IPTG 终浓度0.2~0.8μM 都可以诱导细菌表达蛋白;

b. 加入IPTG 后,把细菌生长温度降至16~30度。较低的生长温度降低了无活性聚集体形成的速率和疏水相互作用,从而可减少包涵体的形成;

c. 添加可促进重组蛋白质可溶性表达的添加剂,培养E.coli 时添加高浓度的多醇类、蔗糖或非代谢糖可以阻止分泌到周质的蛋白质聚集反应,在最适浓度范围内添加这些添加剂不会影响细胞的生长、蛋白质的合成或运输,其它添加剂还有乙醇(诱导热休克蛋白的表达)、低分子量的巯基或二硫化合物(影响细胞周质的还原态,从而影响二硫键的形成)和NaCl ;

d. 供给丰富的培养基,优化培养条件,如供氧、pH 等。

3. 超声后加入终浓度1% Triton x-100处理30~60min ,有利于去除膜碎片和膜蛋白;

4. 若需表达的蛋白含稀有密码子较多,尝试更换E.c oli 宿主菌株,如Rosseta 。

5. 若表达毒性蛋白,造成细菌死亡或者生长缓慢,可以使用pLysS 菌株。

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